This invention relates to a process for producing yeast capable of heat-regulated synthesis of selected and fused proteins. More particularly, this invention relates to a process for cloning selected genes coupled to yeast heat shock inducible genes to produce yeast capable of heat-regulated synthesis of selected and fused proteins.
By the use of recombinant DNA technology it is, in principle, possible to introduce any given DNA sequence into an organism. The method for achieving such a DNA transfer generally employs a procedure which joins the selected DNA to a suitable DNA molecule which may be introduced into the desired organism by transformation. The suitable DNA (termed transfer vector) will contain sequences which allow for the selection of transformants by being able to correct a particular genetic defect in the host organism or by providing resistance to certain drugs. Additionally, the suitable DNA contains sequences which allow this molecule to replicate autonomously in the desired organism or to be integrated into one (or more) of the chromosomes of the desired organism.
If the given DNA sequence encodes a given protein, in order for this protein to be expressed in an organism a number of events must first occur. The DNA sequence must be transcribed by the recipient organism to give rise to an RNA molecule which contains all of the protein coding sequences. This RNA transcript must then attach to the ribosomes of the recipient cell in order for translation to occur. In order for the protein to be properly translated, translation must begin exactly at the nucleotide sequence which encodes the initiation of protein synthesis. Once this has occurred, the universality of the genetic code will allow protein synthesis to proceed to synthesize the desired protein product.
While the genetic code is universal, the DNA sequences which control the site of transcription origin and the regulation of this transcription (termed regulatory sequences or promotor sequences) are specific for given genes in a given organism. Therefore, the sequences which regulate transcription in one organism may not have this function in another organism. Furthermore, the efficiency with which a given RNA is translated contains organism-specific sequences as well. Thus, merely because a DNA sequence is transcribed to give rise to an RNA, this by no means ensures that this RNA will be properly translated. The position of the sequence encoding the origin of translation must be appropriately situated relative to the ends of the RNA molecule in order for translation to give rise to the proper protein product.
To allow for the expression of a given selected DNA sequence in a desired host organism, the regulatory sequences and sequences necessary for ribosome binding obtained from a gene known to function in the host organism must be placed in appropriate position relative to the selected DNA sequence. If the gene fusion is such that only coding sequences of the selected DNA are utilized, the result may be the production of the selected protein. If the fusion is such that in addition to the promoter sequences and ribosome binding sites a portion of the coding sequence for the host cell protein is placed adjacent to the coding sequence for the selected protein (and the fusion is such that the translational reading frame established by the "upstream" host protein sequences is the same as that employed by the "downstream" selected protein coding sequences), the result is a fused protein whose amino end consists of host protein and whose carboxyl end consists of selected protein.
Commercially valuable proteins, such as enzymes, hormones, and proteins utilized in the production of vaccines, may be synthesized in yeast cells which have been modified by recombinant DNA technology as described above. See Hitzemen et al., "Expression of a Human Gene for Interferon in Yeast," Nature 293, 713 (1981); Valenzuela et al., "Synthesis and Assembly of Hepatitis B Virus Surface Antigen Particles in Yeast," Nature 298, 347 (1982). The economic value of such genetic engineering is substantial as yeast may be propagated rapidly and cultured in large scale fermentors.
Current methods for the regulation of gene expression and protein synthesis within genetically modified yeast cells require a change in the nutritional status of the culture medium, such as the addition or removal of a particular nutrient. For example, utilization of the inducible yeast phosphatase gene to regulate the expression of a selected gene requires that cultures have low levels of inorganic phosphate as the gene is unexpressed in the presence of high levels of inorganic phosphate. Therefore, regulating the synthesis of a selected protein using the inducible yeast phosphatase gene requires an appropriate means to remove phosphate from the growth medium. Such a nutritional change in the culture medium is impractical. Alternatively, the gene for galactokinase may be employed to allow regulated expression of a selected gene. In this instance, glucose must be absent from the growth medium and gene expression is initiated upon addition of galactose. However, this method is very costly as galactose is an expensive substrate. Alternatively, the genes encoding for a number of enzymes of glycolysis may be employed to allow the expression of selected genes in yeast. However, this method is also impractical as these genes are not readily regulated. Thus, where regulated expression in yeast can occur, it requires an impractical or expensive alteration in the nutritional status of the culture medium, considerably diminishing the value of synthesizing desirable proteins within yeast.
The present invention overcomes the limitations discussed above by utilizing heat shock inducible genes of an appropriate yeast microorganism. Expression of heat shock inducible genes may be induced merely by raising the cultivation temperature of the yeast microorganism. Thus, the manipulation of temperature elevation will induce the synthesis of yeast heat shock proteins when the yeast organism is cultivated in any medium which allows cell growth. Furthermore, the heat shock response, elicited by a shift in the cultivation temperature beyond a critical level, results in a dramatic alteration of cellular transcription and ultimately in an increased level synthesis of heat shock protein. See, McAlister et al., "Altered Patterns of Protein Synthesis Induced by Heat Shock of Yeast," Current Genetics 1, 63 (1979).
Accordingly, the present invention provides a means for heat-regulated expression of a selected gene in a host yeast by fusion of the selected gene with the regulatory sequence of a yeast heat shock inducible gene. Thus, the point in cultivation where selected and fused proteins are synthesized may be controlled by a simple thermal shift. Further, fusion of a selected gene to the regulatory sequence of the heat shock inducible gene permits a high level of production of selected and fused proteins in a yeast host for the duration of the heat shock response.